Modulation of tRNAAla identity by inorganic pyrophosphatase

Abstract

A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3'-(32)P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA(Ala). The effect of tRNA(Ala) identity mutations on both aminoacylation efficiency (k(cat)/K(M)) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the k(cat)/K(M) value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNA(Ala) identity.

Figure 1

Time course of YFA2 aminoacylation by E. coli AlaRS measured by our TLC assay or the traditional incorporation of [³H]Ala. Aminoacylation was performed at 1 μM tRNA, 20 μM Ala, and 5 (A) or 10 nM AlaRS (B). (A) Time course of 3′-³²P-labeled YFA2 aminoacylation analyzed by TLC. (B) Aminoacylation measured in a double-label experiment either by TLC (●) or ³H incorporation (○).

Figure 2

Effect of PPase on tRNA aminoacylation by E. coli AlaRS. (A and B) Aminoacylation was performed at 1 μM YFA2 and 0.25 (▵), 2.5 (⋄), 25 (□), or 250 (○) nM AlaRS. Open symbols, no PPase added; closed symbols, in the presence of PPase. (C) Time course of YFA2 aminoacylation in the absence of PPase (○). Aminoacylation was performed at 100 nM AlaRS and 1 μM tRNA. After 10 min, 10 units/ml PPase (♦) or 100 nM AlaRS (□) were added.